Method for treating established spontaneous auto-immune diseases in mammals

ABSTRACT

A method of treating spontaneous and ongoing auto-immune diseases in mammals, comprising administering to a mammal, in need of such a treatment, a therapeutically effective amount of one or more non mitogenic anti-CD3 active principles to achieve permanent disease remission through the induction of antigen-specific unresponsiveness, i.e. immune tolerance.

The invention relates to a method for treating established and ongoingspontaneous auto-immune diseases in mammals.

In order to suppress T cell function, immunotherapy based on the use ofantibodies directed at T cell surface receptors, particularly ofmonoclonal antibodies (mAbs), has been extensively investigated.Particularly, mAbs directed against the CD3 complex of the T cellreceptor have been shown to cause transient T-cell depletion andantigenic modulation of the CD3-T cell receptor complex.

In PNAS USA, vol. 91, p 123-127, 1994 Immunology, the inventors, withother co-authors, have reported that a short term treatment with lowdoses of an anti-CD3 mAb could restore self tolerance toβ-cell-associated antigens, thus inducing complete and durable remissionof the spontaneous auto-immune diabetes, in overtly diabetic NOD (nonobese diabetic) mice.

By further investigating the mode of action of anti-CD3 mAb in thismodel, the inventors have found that the long term effect was obtainedonly when treating animal(s)at a very advanced disease stage, i.e. overtauto-immunity. They also demonstrate that non mitogenic, F(ab′)₂fragments of the entire CD3 mAb, that are much better tolerated than thewhole entire CD3-mAb, also afford a long term in vivo effect in overtlydiabetic NOD mice as did the whole anti-CD3 mAb.

This finding is an unexpected extension of the published data whichuntil now, in both transplantation and antigen and/or pharmacologicallyinduced auto-immunity, has proposed (Fab′)₂ fragments of anti-CD3 mAb aseffective tools to only achieve immunosuppression (an overall depressionof immune responses that is only maintained through the chronicadministration of the drugs), but not to promote permanentantigen-specific unresponsiveness namely, a state of immune tolerance(an antigen-specific immune unresponsiveness that is maintained in theabsence of chronic generalized immunosuppression.

Such results are useful for application to other auto-immune situationswhere similar immunoregulatory mechanisms, such as those present inauto-immune diabetes, have been observed.

Accordingly, an object of the invention is to provide a method oftreatment of spontaneous and ongoing of auto-immune diseases in mammalsto achieve permanent Ag-specific unresponsiveness, without the morbidityand with a minimal humoral response as that encountered whenadministering the whole mitogenic antibody.

Another object of the invention is to provide effective tools useful forsuch a method of treatment.

According to the invention, the method of treating auto-immune diseasesin mammals comprises administering to a mammal, in need of such atreatment, a therapeutically effective amount of one or more nonmitogenic anti-CD3 active principles to achieve permanent diseaseremission through the induction of antigen-specific unresponsiveness,i.e. immune tolerance.

Such a treatment was shown to be able to promote durable remission ofthe established disease without the clinical side effects involved whenadministering mitogenic whole anti-CD3 antibodies.

Particularly preferred non mitogenic anti-CD3 antibodies are monoclonalantibodies or fragments thereof, especially F(ab′)₂ fragments.

Said fragments are advantageously such as obtained by pepsin digestionof the whole antibody.

In view of their therapeutical use, said active principle(s) are highlypurified and particularly endotoxin-free.

Said non mitogenic anti-CD3 monoclonal antibody, or fragment thereof isof murine origin or is an humanized antibody.

The permanent antigen-specific unresponsiveness obtained with saidanti-CD3 active principles make them particularly useful as therapeutictools for treating auto-immune pathologies. In particular, they aresuitable for treating diabetes, rheumatoid arthritis, multiple sclerosisor psoriasis.

In said applications, they will be administered, if desired, incombination with other active ingredients and/or compounds whichfacilitate their assimilation.

Said therapeutical tools are advantageously administered in combinationwith pharmaceutical carriers under the form of pharmaceuticalpreparations.

Different forms of administration may be used, especially for injectableroute.

The injectable forms contain 5 to 20 mg of active principle per unitdose, preferably from 5 to 10 mg.

For information only, the dose which can be used in the treatment ofauto-immune diseases in humans, for example diabetes, is 5 to 10 mg/dayfor 10 to 14 consecutive doses.

The invention now will be described with respect to the drawings,wherein:

FIG. 1A shows the kinetics of expression of Interleukin-2 (IL-2),Interleukin-4 (IL-4), and β-2 microglobulin (β-2m) RNA in NOD femalemice that were given intact CD3 monoclonal antibody, F(ab′)₂ fragmentsof CD3 monoclonal antibody, L2 antibody, or saline; and

FIG. 1B shows the results of semiquantification of amplificationproducts using doubling dilutions of cDNA for PCR reactions for IL-2,IL4, and IL-10.

Other characteristics and advantages of the invention will appear fromthe examples given hereinbelow.

EXAMPLE 1

Treatment of overtly diabetic NOD female mice with purified F(ab′)₂fragments of CD3 anti-mAb.

NOD mice (K^(d), I-A^(NOD), D^(b)) were bred under specificpathogen-free conditions; in females, spontaneous IDDM appears by 14weeks of age (90% incidence at 30 weeks of age) and is preceded byinsulitis at 4 to 6 weeks.

In a preferred embodiment of the invention said non mitogenic anti-CD3active principle is a non mitogenic anti-CD3 antibody or a fragmentthereof. Such fragments are advantageously F(ab′)₂ fragments.

The cell line producing the hamster 1452C11 mAb (IgG, anti-mouse CD3ε-chain) was used in those experiments (O. M. Les et al, 1987, PNAS USA84:1274)

The anti-CD3 mAb F(ab′)₂ fragments were prepared by pepsin digestion.

Pepsin (Sigma Chemical Co., St. Louis, Mo.) was used at a finalconcentration of 2% (20 μg/mg of purified antibody) in 1M acetatebuffer, pH 3. Digestion was conducted for 2 h at 37° C. Followingdialysis at 4° C. using 0.1 M PBS pH 8, digested F(ab′)₂ fragments werepurified in two steps : a protein A-Sepharose CL-4B affinitychromatography column to eliminate digested Fc fragments, then gelfiltration of the nonretained fraction on an Ultragel AcA54 column(Pharmacia, Uppsala, Sweden).

The physico-chemical properties of the fragment preparations wereanalyzed by SDS-PAGE.

The binding capacity was tested by immunofluorescence in a classicalcompetition assay using purified FITC-labeled whole CD3 mAb.

The digestion and purification of F(ab′)₂ fragments was performed withspecial caution to avoid endotoxin contamination. The material used forin vivo treatment was negative in the Limulus assay.

NOD females presenting with overt diabetes were included in thetreatment protocol when a fasting glycemia ranging 3.5 to 4 g/L wasscored on two consecutive occasions. Mice were then randomized toreceive a treatment with CD3 mAb F(ab′)₂ fragments (50 μg/day for 5consecutive days), whole CD3 mAb (5-20 μg/day for 5 consecutive days),or as a control normal hamster Igs.

Complete remission was defined as a return to normal glycemia and thedisappearance of glycosuria in the absence of any exogenous insulinsupply. Histopathology on paraffin sections of Bovin-fixed or frozenpancreatic tissue were performed as previously described (1). Scoring ofmononuclear cell infiltration was as follows: grade 0=normal islets;grade 1=focal or peripheral insulitis (lymphocytes around the islet, butno destruction of endocrine cells as assessed by labeling withanti-insulin Abs); and grade 2 invasive destructive insulitis.

The results regarding the remission of overt diabetes in the micefollowing the short treatment with purified F(ab′)₂ fragements are givenin Table 1. TABLE I % Remission of IDDM Weeks After Anti-CD3 F(ab′)₃Hamster 1 g Treatment n = 42 n − 18 0 0 0 2 55 22 4 62 16 6 64 0 10 67 020 67 0

The difference in percent remission between CD3 mAB F(ab′)₂fragements-treated and control animals is statistically significant(p<0.0l) using χ² test.

As shown by said results, F(ab′)₂ fragments of the mAb appeared potentin promoting permanent remission of overt diabetes in the conditions ofthe experiments.

EXAMPLE 2

Study of the triggering effect of F(ab′)₂ fragements of CD3 mAb oncytokine gene transcription.

NOD females received a single i.v. injection of either intact 145 2C11CD3 mAb (20 μg) or purified F(ab′)₂ fragments of 145 2C11 CD3 mAb (50μg). Mice injected with saline or with 5 to 50 pg of L2, a hamster mAbspecific for recombinant but not natural mouse IL-2, were used ascontrols. Three individual animals were analyzed in each group. Spleencells were collected before any treatment and at various times followinginjection of the different preparations, and RNA was extracted forRT-PCR.

Crude RNA was extracted using TRIzol (Life Technologies) followed byisopropanol precipitation. For reverse transcription (RT), total RNA (6μl in a final volume of 12 μl) was added to 18 μl of cDNA synthesisreaction mixture. Two microliters of RT product was amplified using PCRfor 30 cycles, final volume of 50 μl, with standard buffer conditionsand a final Mg²⁺ concentration of 1.5 mM (2.5 U Taq UNA polymerase, LifeTechnologies) Each PCR cycle consisted of 1 min. at 94° C., 1 min. at55° C., and 1 min. at 72° C. on a Techne thermal cycler (Osi, Paris,France); 100 ng of cDNA was used for PCR unless stated otherwise. Whenneeded to semi-quantitate the amplification products obtained, PCR withdoubling dilutions of cDNA was performed. The following primers(Bioprobe Systems, France) were used IFN-γ 5′ primer, CCA GCA GAG AATGGA AAG TC; IFN-γ 3′ primer. GAT GCT GCT TAC ATG TCT CG; IL.2 5′ primerCCA GCA GAG AAT GGA AAG TC; IL-2 3′ primer, GAT GCT GCT TAC ATG TCT CG;IL-4 5′ primer. TCG GCA TTT TGA ACG AGG TC, IL-4 3′ primer. GAA AAG CCCGAA AGA GTC TC; IL-10 5′ primer, GGG ATG ACA GTA GGG GAA CC; IL-10 3′primer, AGA GCA AGG CAG TGG AGC AG: β₂-microglobulin 5′ primer, CCA GCAGAG AAT GGA AAG TC β₂-microglobulin 3′ primer, GAT GCT GCT TAC ATG TCTCG. Ten microliters of RT-PCR products were separated by 1.2% agarosegel electrophoresis in 1×TBE (Tris-borate-EDTA) containing 0.2 μg/ml ofethidium bromide and visualized under UV light. Where products weresemiquantified, RT-PCR, β₂ microglobulin mRNA was used as a housekeepingreporter gene.

The kinetics of mRNA expression is shown in FIG. 1, part A.Semiquantification of amplification products was performed usingdoubling dilutions of cDNA for PCR reactions; data are shown for IL-2,IL-4, and IL-10 in FIG. 1, part B.

As shown in FIG. 1A, using PCR on splenocytes from anti-CD3F(ab′)₂-treated animals, enables identification of the transcription ofmRNAs specific for IL-2, IL-4, IL-10, and IFN-γ. Semiquantificationusing serial dilution of cDNA samples suggested that, as compared withwhat was observed in NOD mice treated with intact CD3 mAb, F(ab′)₂fragments promote a less effective transcription of IL-2 mRNA, whereassimilar levels of IL-4, IL-10, and IFN-γ message were detected (FIG.1B).

EXAMPLE 3

Cytokine production by stimulated spleen cells from CD3 mAb- andF(ab′)₂-treated NOD mice.

Spleen cells from CD3 mAb- and F(ab′)₂-treated NOD mice were collectedat different times after treatment and tested for their capacity tosecrete IFN-γ and IL-4 upon mitogenic stimulation using Con A.

Spleen cells from CD3 or F(ab′)₂ fragments-treated animals werecollected and cultured in vitro (1×10°/ml) in 24-well plates, for 24 to48 h in a humidified atmosphere containing 5% CO₂, in DMEM-Glu-tamax(Life Technologies, Paisley, Scotland) supplemented with 100 IU/mlpenicillin, 100 μg/ml streptomycin, sodium pyruvate, nonessentialaminoacids, 0.05 mM β-mercaptoethanol, and 10% FCS. In stimulatedcultures, Con A was added at a final concentration of 10 μg/ml.Supernatants were collected and stored frozen at −80° C. until tested.IFN-γ and IL-4 were quantitated using specific ELISA as alreadydescribed (17). The Abs used for detection were AN18 (kindly provided byDr. A. O'Garra, DNAX, Palo Alto, Calif.) and biotinylated R46A2 forIFN-γ and 11B11 and biotinylated BVD6 (kindly provided by A. O'Garra)for IL-4, Mouse rIL-4 (R&D Systems. Minneapolis, Minn.) and IFN-γ wereused as internal standards. Detection limits were 0.2 ng/ml for IL-4 and0.1 ng/ml for IFN-γ.

The results are given in Table 2 TABLE 2 Time from Treatment Treatment(week) IFN-γ (ng/ml) Anti-CD3 2  68.36 ± 11.51* Anti-CD3 F(ab′)₂ 2 16.37 ± 3.40* Hamster Ig 2  101.90 ± 13.34* Anti-CD3 7  31.74 ± 4.14⁵Anti-CD3 F(ab′)₂ 7  23.21 ± 5.69¹ Hamster Ig 7 35.76 ± 4.20 Untreatedcontrols 29.42 ± 7.11

As compared with age-matched controls injected with irrelevant hamsterIg, polyclonally activated spleen cells from CD3 mAb- and F(ab′)₂fragment-related NOD mice showed, for about 5 wk from the end oftreatment, a significantly decreased IFN-γ-producing ability (Table II).

EXAMPLE 4

Pharmaceutical formulation

The active principles are formulated under a desaggregated form andeither lyophilyzed or suspended into an appropriate liquid, each dosecontaining, as above mentioned, 5 to 20 mg of non mitogenic antibody ora fragment thereof.

1-15. (canceled)
 16. A method of treating an ongoing autoimmune diseasein a human having said disease, wherein said disease is psoriasis,comprising: treating said human by administering one or morenon-mitogenic anti-CD3 active compounds selected from the groupconsisting of CD3 antibodies and fragments of CD3 antibodies in anamount effective to treat said psoriasis.
 17. The method of claim 16wherein said treatment results in a durable remission of said psoriasis.18. The method of claim 16 wherein said administration of said one ormore non-mitogenic anti-CD3 active compounds is a non-chronicadministration.
 19. The method of claim 16 wherein said non-mitogenicanti-CD3 active compound is a non-mitogenic anti-CD3 monoclonal antibody(Fab′)₂ fragment.
 20. The method of claim 16 wherein said non-mitogenicanti-CD3 active compound is a non-mitogenic anti-CD3 antibody.
 21. Themethod of claim 20 wherein said non-mitogenic anti-CD3 active compoundis a non-mitogenic anti-CD3 monoclonal antibody.
 22. The method of claim21 wherein said monoclonal antibody is selected from the groupconsisting of murine or humanized antibody.
 23. The method of claim 16wherein said compound is highly purified and endotoxin-free.
 24. Themethod of claim 16 wherein said active compound is administered byinjection.
 25. The method of claim 16 wherein said non-mitogenicanti-CD3 active compound is administered in an injectable form thatcontains from 5 to 20 mg of the non-mitogenic anti-CD3 active compound.